Engineered lumenized vascular networks and support matrix

ABSTRACT

Disclosed herein are capillary fabrication devices comprising living cells within a support medium. Culture of the cells produces viable lumenized capillary networks with natural or pre-determined geometries and ECM and basement membrane associated with the capillary networks. The capillary networks and the ECM and basement membrane detachable from the capillary networks are useful for tissue engineering applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional under 35 U.S.C. § 120 of U.S.application Ser. No. 13/635,043, filed Sep. 14, 2012, which is a U.S.national counterpart application of international application serial No.PCT/US2011/028492, filed Mar. 15, 2011, which claims the benefit of U.S.Provisional Patent Application No. 61/313,886, filed Mar. 15, 2010. Theentire disclosures of U.S. patent application Ser. No. 13/635,043,PCT/US2011/028492, and U.S. Ser. No. 61/313,886 are hereby incorporatedby reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM076692 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

FIELD

This disclosure generally relates to the field of biomedical tissueengineering. More specifically, the disclosure relates to devices forthe synthesis of capillary networks with natural or controlledgeometries and their associated extracellular matrix and basementmembrane.

BACKGROUND AND SUMMARY

Under the proper conditions, endothelial cells or theirprecursors/progenitor cells, spontaneously assemble into networks anddifferentiate to form lumenized connected capillary networks.

Both the forming capillary network and the final capillary network andits associated extracellular matrix (ECM) have numerous scientific,industrial and medical applications. In vitro angiogenesis assaysemploying the forming capillary network (or capillary fabricationdevices) play a crucial role in identifying factors involved in vasculardevelopment. Such assays are used in drug development asmoderate-throughput screens for angiogenesis promoters and inhibitorsrelated to wound healing, age-related macular degeneration, diabetes,cancer, and other diseases.

The completed networks formed by in vitro angiogenesis devices are alsoimportant in tissue engineering, as vascular replacements for humanimplantation, while the ECM of the networks can be employed as ascaffold implanted into patients to promote the growth of the patient'sown vasculature into the pattern provided by the ECM.

Drug development and tissue engineering applications both require rapidcreation of viable lumenized capillary networks having properties asclose as possible to in vivo capillary networks. Existing in vitroangiogenesis techniques are not capable of rapidly creating lumenizedcapillary networks with properties substantially similar to in vivonetworks.

Current methods of tissue engineering are limited to relatively thinand/or avascular tissues like skin, cartilage, and bladder, wherepost-implantation vascularization from the host is sufficient to meetthe implant's demand for oxygen and nutrients. Vascularization remains acritical obstacle for engineering thicker, metabolically demandingorgans, such as heart and liver. Thus the survival of tissue-engineeredthree-dimensional constructs in vivo depends on providing enough bloodsupply to the engineered tissue and the capacity of the engineeredmicrocirculation to connect with the existing circulation of therecipient.

Engineering thicker, metabolically demanding organs, such as heart andliver, requires techniques for manufacturing microvessels with highlycontrolled geometries. Existing microcirculation engineering techniquesare not capable of constructing controlled-geometry microvessels capableof connection to the host vessels, expansion of vascular volumeaccompanying growing tissue, and prevention of excessive vascularregression.

Current in vitro capillary fabrication devices are eitherquasi-two-dimensional or three-dimensional. Quasi-two-dimensionaldevices are categorized as either rapid or long-term. Rapidquasi-two-dimensional devices consist of a layer of endothelial cellsseeded sub-confluently on top of a thick layer of a basement-membranegel which is made of a mixture of collagen, fibrin, or Matrigel™.Depending on the components and mechanical properties of the gel,endothelial cells align to form a one-cell thick capillary-like patternwithin 1 to 3 days. In rapid devices, the standard Matrigel™ capillaryfabrication device (FIG. 4) is widely used, especially in assayingapplications to characterize anti-angiogenic or pro-angiogenic factors.In the standard quasi-two-dimensional Matrigel™ capillary fabricationdevice, cells plated on a thick layer (about 0.5 mm) of Matrigel™ areexposed to high levels of soluble and ECM-bound growth factors. A highconcentration of growth factors in Matrigel™ (or even Growth FactorReduced (GFR) Matrigel™) results in artifacts and over-stimulation ofcells. Further, cell motility is restricted by adhesion of cells to thesolid Matrigel™ and resultant capillaries have endothelial cells thatare abnormally elongated compared to the in vivo morphology ofendothelial cells. The endothelial cells typically die 24 to 48 hoursafter forming the networks in rapid quasi-two-dimensional devices (Rantaet al. (1998), J Cell Physiol. 176(1):92-98; Vailhé et al. (2001) LabInvest. 81(4):439-452); thus these devices are not suitable forapplications requiring viable capillary networks. However, the lumenizedcapillary networks formed in the devices disclosed herein remain viablefor about 4 weeks.

Long-term quasi-two-dimensional devices generally consist of endothelialcells suspended in normal culture medium and conditions withoutinclusion of an extracellular matrix substrate. As the endothelial cellsdivide, they form a confluent mono-layer, after which some differentiatespontaneously to form capillary-like structures on top of a confluentlayer of undifferentiated endothelial cells. Long-term devices require 2to 8 weeks of cell culture, making them unsuitable for high-throughputexperiments.

Applications of quasi-two-dimensional angiogenesis devices include, forexample, studies of the role and synthesis of extracellular matrix invascular morphogenesis; studies of the roles of adhesion molecules;screening of angiostatic molecules; functional characterization ofendothelial cell lines; studies of proteases; extracellular proteinsynthesis; vessel maturation; and studies of the role of glycationproducts in diabetes.

Three-dimensional capillary fabrication devices combine endothelialcells with a three-dimensional gel which the endothelial cells theninvade. Widely-used three-dimensional in vitro devices include aorticrings in gelified matrices, endothelial cells seeded inside a gel orsandwiched between two layers of gel or between a single layer of geland a cell-culture surface, and microcarrier beads coated withendothelial cells. The forming capillaries in three-dimensionalangiogenesis devices are applied to study the effects of cytokines,metalloproteases and the fibrinolytic pathway during tubulogenesis,endothelial apoptosis, the importance of the configuration andcomposition of the substrate, the role of cell-adhesion molecules, theeffect of hypoxia on endothelial cells, and for screening of angiogenicand angiostatic molecules.

Three-dimensional capillary fabrication devices require many more cellsto achieve the cell densities required for fabrication of connectedcapillary networks. Cell distribution in three-dimensional capillaryfabrication devices is not uniform which may cause over-crowded regionsand increased cell death. The transport of oxygen and other nutrients tothree-dimensional matrices via diffusion limits the thickness of thethree-dimensional matrices in those capillary fabrication devices. Thecell resources in capillary fabrication devices are prohibitivelylimited and expensive. Since the cells are embedded in a solidified gelwhich limits cell motility, the formation of capillary-like patterns isslow, taking 1 to 8 weeks. Thus three-dimensional capillary fabricationdevices are inefficient and slow which make them not suitable forindustrial, drug-development and high throughput applications.

Thus, improvements are needed for in vitro capillary fabricationdevices.

Techniques for the vascularization of tissue-engineered constructs canbe broadly grouped into in vitro and in vivo approaches. In vivoapproaches rely on vessel ingrowth from host to the engineered tissue.This ingrowth process is often limited to tenths of microns per days,meaning that the time needed for complete vascularization of an implantof several millimeters is in the order of weeks. Several strategies havebeen developed for improving the ingrowth of vessels from host tissueincluding scaffold designs, angiogenic-factor delivery and in vivopre-vascularization. In vitro approaches include in vitropre-vascularization techniques. In vitro approaches do not rely oningrowth of host vessels into entire construct. However, anastomosis ofthe vessels in the in vitro-pre-vascularized tissues is not as fast asin vivo pre-vascularization. In vitro-pre-vascularization also needs tohave proper organization and geometry to be able to provide enough bloodperfusion after implantation of the engineered tissue.

The capillary fabrication devices disclosed herein can produce highlycontrolled functional lumenized capillary networks that enhance bloodperfusion in the engineered tissue.

Disclosed herein are capillary fabrication devices for manufacturingforming capillary networks and formed capillary networks with natural orcontrolled geometries the engineered ECM and basement membraneassociated with the capillary networks, and application of the capillarynetworks and ECM and basement membrane for construction of engineeredtissues.

The capillary fabrication devices described herein produce capillarieswhich improve in a number of ways on the standard quasi-two-dimensionalMatrigel™ capillary fabrication device. Unlike the standardquasi-two-dimensional Matrigel™ capillary fabrication device, whichproduces a capillary-like structure composed of unnaturally elongatedendothelial cells, the morphology of endothelial cells incorporated inthe capillary cords and resulting lumens in the capillary fabricationdevice described herein are very similar to those in capillaries formedin vivo, e.g., in zebrafish embryos and chick allantois.

It has also been found that the standard quasi-two-dimensional Matrigel™capillary fabrication device (even when growth-factor reduced Matrigel™is used) produces a honeycomb-like network, in which tubules are oftencomposed of a single endothelial cell stretched and connected toaggregates of endothelial cells. Most capillary tubules formed in thecapillary fabrication devices described herein are composed of severalcell lengths, and are thus capable of producing mean tubule lengthscomparable to capillaries in vivo (FIG. 1). Capillary networks producedusing the standard quasi-two-dimensional Matrigel™ capillary fabricationdevice are not viable for more than 48 hours. The lumenized capillarynetworks of the capillary fabrication devices described herein may beviable for up to 4 weeks after formation of the network.

The standard quasi-two-dimensional Matrigel™ capillary fabricationdevice is unable to recapitulate normal cell motility and proliferationsince endothelial cells plated on top of a thick layer of Matrigel™ showno or little proliferation and motility. The cells do not haveproperties that are substantially similar to in vivo cells. For example,mean tubule diameter which has significant biological importance, isusually less than 5 microns in the standard quasi-two-dimensionalMatrigel™ capillary fabrication device. Formation of narrow tubes (innerdiameter<4 microns) in the standard quasi-two-dimensional Matrigel™capillary fabrication device also indicates high levels of mechanicalstress in the individual cells. Tubes less than 4 microns in diameter donot support natural blood flow. Thus they are non-functional. However,the diameter of engineered lumenized capillaries produced using thecapillary fabrication devices described herein were found to range from5 to 20 microns, which matches the capillary diameters of zebrafishembryos, chick allantois and many human tissues.

In the capillary fabrication devices described herein, only endotheliallineage cells incorporated in capillary cords are not motile and showreduced proliferation potency because of strong junctional complexes;the rest of the cells proliferate and are motile. Thus, the capillaryfabrication devices described herein are less sensitive to the seededcell density than the standard quasi-two-dimensional Matrigel™ capillaryfabrication device. This allows formation of capillary networks fromlimited numbers of stem cells or from an autograft. An autograftcapillary network has a lower chance of rejection in tissue repair andengineering applications.

In the capillary fabrication devices described herein, similar tolong-term in vitro devices, the lumenized capillary networks remainviable for at least one week after tubulogenesis, and often remainviable for four weeks or more. In contrast, in most in vitro capillaryfabrication devices, the capillary-like networks degrade and disappearafter tubulogenesis. The longer viability of the engineered capillarynetworks described herein allows for assays to study the effects ofpro/anti-angiogenic factors on both established lumenized capillarynetworks and the initial stages of tubulogenesis. The steps ofendothelial-cell tubulogenesis can be recapitulated in quasi-in vivoconditions.

Use of cell-culture-treated dishes, rather than non-treated dishes, doesnot produce a capillary-like pattern, but rather increases proliferationof seeded endothelial resulting in a confluent layer of undifferentiatedendothelial cells. Typically, normally adherent cells cultured innon-treated polystyrene dishes without coating undergo anoikis/apoptosisafter a few hours and die. However, in the capillary fabrication devicedisclosed herein, the use of a support-generating medium to form thesupport medium on a non-treated polystyrene surface as a cell-culturesurface allows the cells to survive and form capillary networks. Withoutbeing bound by theory, it is believed that the binding of specificintegrin receptors in the endothelial cells contribute to the initiationof transcription of anti-apoptotic genes, differentiation andtubulogenesis.

Current methods of tissue engineering are limited by the difficulties offorming functional vascular networks in the engineered tissues.Engineered blood vessels using prosthetic conduits are not suitable forvessel diameters of less than 6 mm due to formation of thromboses.Engineered blood vessels which have a lining of endothelial cells, usingcell-sheet engineering or bioprinting, are limited to large bloodvessels. Thus existing tissue engineering methods are unable to producefunctional vascular networks for tissue engineering. The capillaryfabrication device and custom-patterned capillary fabrication device arecapable of producing functional capillaries which can be integrated withlarger vessels in the host tissue or engineered tissue to formfunctional vascular networks. Engineering thicker metabolicallydemanding organs, such as heart and liver requires techniques formanufacturing microvessels with highly controlled geometries. Existingmicrocirculation engineering techniques are not capable of constructingcontrolled-geometry microvessels capable of connecting to the hostvessels, expansion of vascular volume accompanying growing tissue, andprevention of excessive vascular regression. Thecustom-patterned/controlled-geometry capillary device is capable ofproducing functional lumenized capillary networks which can highlyoptimize blood perfusion in the engineered tissue.

In medically-oriented tissue engineering applications autologousresources of cells are limited. Thus engineering tissues from autologouscomponents is often not practical. Extracellular components are highlyconserved and known to enhance and regulate growth/regrowth anddifferentiation of cells in engineered tissues. Use oforganically-fabricated ECM and basement membrane (formed using thedevices disclosed herein) of non-autologous or non-human (like mouse)origin can be useful for medical tissue-repair and tissue engineeringapplications with minimal transfer of external factors and thussignificant reduction of the likelihood of rejection after implant.

The following various embodiments are provided:

1) A device for fabrication of engineered capillary networks comprising:

living cells;

a cell-culture surface; and

a support-generating medium, the support-generating medium comprising agel forming material and a liquid cell-culture medium, wherein the gelforming material is substantially dissolved in the cell-culture mediumand forms a support medium on the cell-culture surface.

2) A custom-patterned capillary fabrication device comprising:

living cells;

a cell-culture surface; and a support-generating medium, thesupport-generating medium comprising a gel forming material and a liquidcell-culture medium, wherein the gel forming material is substantiallydissolved in the cell-culture medium and forms a support medium on thecell-culture surface, and wherein the cell-culture surface comprises anetwork-like pattern containing regions of varying hydrophobicity.

3) The device of any of clauses 1 to 2 wherein the cells are of humanorigin.

4) The device of any of clauses 1 to 3 wherein the living cells are ofendothelial lineage.

5) The device of any of clauses 1 to 4 wherein the cells are selectedfrom the group consisting of embryonic stem cells, endothelialprogenitor cells, circulating endothelial cells, and lymphaticendothelial cells.

6) The device of any of clauses 4 to 5 further comprising at least oneadditional cell type.

7) The device of clause 6 wherein the additional cell type is selectedfrom the group consisting of pericytes, smooth muscle cells,fibroblasts, and any combination thereof.

8) The device of any of clauses 1 to 7 wherein one or more cells aremodified cells.

9) The device of any of clauses 1 to 8, wherein the cell-culture surfacecomprises at least one hydrophobic region.

10) The device of any of clauses 1 to 9 wherein the cell-culture surfacecomprises a coating of at least one temperature sensitive polymer.

11) The device of clause 10 wherein at least one temperature sensitivepolymer is poly(N-isopropylacrylamide).

12) The device of any of clauses 1 to 11 wherein the cell-culturesurface is flat.

13) The device of clause 12 wherein the cell-culture surface is selectedfrom the group consisting of petri dishes, well-plates, slides, andcoverslips.

14) The device of any of clauses 1 to 13 wherein the cell-culturesurface is comprised of a material selected from the group consisting ofnon-treated polystyrene, glass, a temperature sensitive polymer, and anycombination thereof.

15) The device of any of clauses 1 to 14 wherein the cell-culturesurface further includes meshes or scaffolds.

16) The device of any of clauses 1 to 15 wherein the cell-culturesurface is modified by etching, stamping, contact printing, UV laserablation, or any combination thereof.

17) The device of any of clauses 1 to 16 wherein the cell-culture mediumis a defined cell-culture medium.

18) The device of any of clauses 1 to 16 wherein the cell-culture mediumcomprises serum albumin.

19) The device of any of clauses 1 to 18 wherein the cell-culture mediumcomprises a bicarbonate-base or HEPES buffer.

20) The device of any of clauses 1 to 19 wherein the gel-formingmaterial comprises at least one extracellular matrix (ECM) protein.

21) The device of any of clauses 1 to 20 wherein the gel formingmaterial comprises at least one protein selected from the groupconsisting of laminin, collagen IV, heparan sulfate proteoglycans,entactin/nidogen, TGF-β, epidermal growth factor, insulin-like growthfactor, fibroblast growth factor, and tissue plasminogen activator.

22) The device of any of clauses 1 to 20 wherein the gel-formingmaterial is Matrigel™.

23) The device of any of clauses 1 to 20 wherein the support-generatingmedium comprises Matrigel™ diluted from 1 to 30 to about 1 to 60 timesin the liquid cell-culture medium.

24) The device of any of clauses 1 to 20 wherein the support-generatingmedium is dissolved in the liquid-cell culture medium to yield an ECMprotein concentration of from about 170 μg ECM proteins per ml ofliquid-cell culture medium to about 350 μg ECM proteins per ml ofliquid-cell culture medium.

25) The device of any of clauses 1 to 24 wherein the support mediumharbors the living cells.

26) The device of any of clauses 1 to 25 wherein the support medium hasa thickness of less than 150 microns.

27) The device of any of clauses 1 to 25 wherein the support medium hasa thickness of less than 50 microns.

28) The device of any of clauses 1 to 25 wherein the support medium hasa thickness from 50 microns to 100 microns.

29) The device of any of clauses 1 to 25 wherein the support medium hasa thickness from 20 microns to 40 microns.

30) A formed capillary network made using the device according to any ofclauses 1 to 29.

31) The capillary network of clause 30 wherein the capillary networkcomprises tubules having a diameter of at least 5 microns.

32) The capillary network of clause 30 wherein the capillary networkcomprises tubules having a diameter of at least 10 microns.

33) The capillary network of clause 30 wherein the capillary networkcomprises tubules having a diameter of at least 15 microns.

34) The capillary network of clause 30 wherein the capillary networkcomprises tubules having a diameter of at least 20 microns.

35) The capillary network of any of clauses 30 to 34 wherein thecapillary network geometry is pre-patterned.

36) Use of the device according to any of clauses 1 to 29 to generate aformed capillary network.

37) Use of the device of any of clauses 1 to 29 to generate a formingcapillary network.

38) The use according to any of clauses 36 to 37 wherein the livingcells are contacted by a test substance suspected to promote or inhibitangiogenesis.

39) An ECM and basement membrane produced by the living cells of thedevice according to any of clauses 1 to 29, wherein the ECM and basementmembrane are detached from a forming or formed capillary network.

40) Use of the device according to any of clauses 1 to 29 to generate anECM and basement membrane that are detachable from a forming or formedcapillary network.

41) The use according to clause 40 wherein the ECM and basement membraneare detached from the capillary network by at least changing thecell-culture temperature.

42) The use according to any of clauses 40 to 41 wherein the ECM andbasement membrane are detached from the capillary network by at leastchanging the pH of the cell-culture medium.

43) The use according to any of clauses 40 to 42 wherein the ECM andbasement membrane are detached from the capillary network by at leastcontacting the capillary network and ECM and basement membrane with astream of liquid.

44) Use of a capillary network produced by the device of any of clauses1 to 29 to repair or regenerate tissue in vivo.

45) Use of the ECM and basement membrane of clause 39 in an in vitrotissue engineering application.

46) Use of the ECM and basement membrane of clause 39 in a cell-sheetengineering application.

47) Use of the ECM and basement membrane in a bioprinting application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cultured human umbilical vein endothelial cells (HUVECs)after 7 days of culture using the capillary fabrication device. CalceinAM was loaded for fluorescent imaging at 10 μM. White arrows showsegments of a viable lumenized capillary network. A typicalundifferentiated HUVEC is about 30 μm in, vitro. When integrated into atubule, a typical HUVEC is about 50 to 60 μm. The lumenized capillarylengths range 100 μm to 1500 μm. ECFCs also formed capillaries whencultured using the capillary fabrication device for at least 4 days (notshown).

FIG. 2 shows viable lumenized capillary networks formed from HUVECsafter 36 hours in the disclosed capillary fabrication device asdescribed herein. They remained viable 7 days later. White arrows showthe lumen in the disclosed capillary fabrication device as describedherein. The lumenized capillaries have a diameter of 10 to 15 μm. Arrowheads show endothelial cells which have not integrated into thelumenized capillary network. A typical undifferentiated HUVEC is about30 μm in vitro. When integrated into a tubule, a typical HUVEC is about50 to 60 μm. Bar, 50 μm.

FIG. 3 shows the formation of support medium. After covering thecell-culture surface with support-generating medium containing dissolvedgel-forming material, the support-generating medium forms/deposits aloosely-connected support medium. (A), (B), (C), (D) show phase-contrastimages of the support medium at 30 minutes, 3 hours, 6 hours and 12hours after plating. Support medium is the darker patches of material(black arrow-heads in (A) and (B)). Support medium covers the entiresurface after about 6 hours (C). Support medium coverage does not changesignificantly after 6 hours. (D) The support medium after 12 hours. Thegray background is the hydrophobic cell-culture surface. Bar, 20 μm.

FIG. 4 shows components of a standard two-dimensional Matrigel™capillary fabrication device. Matrigel™ solidifies at room temperature.Endothelial cells are plated on top of a layer (300-500 μm) ofsolidified Matrigel™. Cells are covered by about 2 mm of liquid culturemedium. A quasi two-dimensional capillary-like network one cell lengththick forms on top of the solidified Matrigel™.

FIG. 5 shows components of the capillary fabrication device.Support-generating medium which contains a dissolved gel-formingmaterial forms a gel of support medium of about 10 to 30 μm inthickness. Cells are covered by about 2 mm of support-generating medium.Lumenized capillary networks form inside the support medium.

FIG. 6 shows detached ECM generated using a capillary formation device.The ECM was detached from the bottom of a polystyrene dish by making theliquid culture media slightly acidic (pH˜6.0-6.5). Arrows showmanufactured cables. Scale bar˜250 μm.

FIG. 7 shows the formation of capillaries by HUVECs cultured on apolystyrene dish having regions coated with support medium and regionscoated with highly hydrophobic PDMS. HUVECs grew and formed capillarynetworks on the polystyrene coated with support medium but did not growand form networks on regions coated with PDMS. The boundary between PDMSregions and polystyrene (with support medium) regions are demarcated bytwo black lines. The black arrow shows a formed capillary. Scale bar˜250μm.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

For the purposes of promoting an understanding of the principles of thedisclosure, reference will now be made to illustrative embodiments. Itis understood that no limitation to the scope is intended. It is furtherunderstood that any alterations and modifications to the illustratedembodiments and further applications of the principles as may occur toone skilled in the art to which this invention pertains are includedwithin the scope of the disclosure.

As used herein, the phrase “capillary fabrication device” generallyrefers to a cell-culture which provides an environment for manufacturingcapillary-like patterns.

As used herein, the phrase “controlled-geometry capillary fabricationdevice” or “custom-patterned capillary fabrication device” generallyrefers to a cell-culture which provides an environment for manufacturingcapillary-like networks which have a predefined geometry includingtubule lengths, locations and connectivity.

As used herein, the phrase “forming capillary network” generally refersto a group of cells in a capillary fabrication device which areorganizing to form one or more tubules.

As used herein, the phrase “formed capillary network” generally refersto a group of cells in a capillary fabrication device which areorganized into one or more tubules.

As used herein, the phrase “ECM and basement membrane” generally refersto the extracellular matrix proteins, growth factors, and othercomponents that are associated with the forming or formed capillarynetworks. The ECM and basement membrane may be produced and/or modifiedby the cells in the forming or formed capillary network. In general, ECMincludes basement membrane which is a laminar organization ofextracellular proteins (and associated/other components) includingcollagen type IV and laminin.

As used herein, the phrase “cell-culture surface” generally refers to asolid which can physically support cells and support medium, and theresulting capillaries.

As used herein, the phrase “non-treated polystyrene” refers to apolystyrene cell culture surface that has not been pre-treated (i.e.,prior to contact with the support-generating medium) with anadherence-enhancing substance, corona discharge, gas-plasma, or otherlike treatments. Likewise, the phrase “cell-culture-treated” refers tocell-culture surfaces providing an adhesive substrate for cellattachment and survival.

As used herein, “patterned surface” refers to a cell-culture surfacewhich has spatially or temporally varying physical or chemicalproperties including but not limited to hydrophobicity and proteinbinding affinities. A patterned surface may include glass, biocompatibleand biodegradable materials and polymers including but not limited tonon-treated polystyrene and temperature sensitive polymers. A patternedsurface may include coatings of proteins, polymers, or other materials.A patterned surface may also include scaffolds, meshes and other2-dimensional or 3-dimensional structures.

As used herein, the phrase “temperature sensitive polymer” refers to apolymer which changes its hydration state depending on temperature. Atemperature sensitive polymer includes but is not limited topoly(N-isopropylacrylamide).

As used herein, the phrase “gel-forming material” refers to one or morecomponents that are capable of existing in a gelatinous or highviscosity liquid form. A gel-forming material may include one or moreextracellular matrix (ECM) proteins. Extracellular matrix proteinsinclude, but are not limited to, laminin, collagen IV, heparan sulfateproteoglycans, entactin/nidogen, TGF-β, epidermal growth factor,insulin-like growth factor, fibroblast growth factor, and tissueplasminogen activator, and the like. A gel-forming material ofextracellular matrix proteins includes but is not limited to Matrigel™.

As used herein, “Matrigel™” refers to a commercially availablegelatinous extracellular protein mixture secreted byEngelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Such proteins includelaminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen,TGF-β, epidermal growth factor, insulin-like growth factor, fibroblastgrowth factor, and tissue plasminogen activator, and additionalproteins. Stock Matrigel™ commercially available currently has about 13mg/ml of protein. Approximately, more than 80 percent of Matrigel™ isECM proteins. The concentration of ECM proteins is initially about 10.4mg/ml in Matrigel™ (˜80% of 13 mg). Therefore, Matrigel™ stock diluted1:30 in cell-culture medium (support-generating medium) contains about347 μg of ECM proteins per ml, and a solution diluted 1:60 containsabout 173 μg ECM proteins per ml.

As used herein, “Growth Factor Reduced Matrigel™” (GFR Matrigel™) refersto a commercially available gel that is purified and characterized to agreater extent than standard Matrigel™. GFR Matrigel™ effectively haslower levels of a variety of growth factors except for TGF-beta whichmay be bound to collagen IV and/or sequestered in a latent form thatpartitions with the major components in the purification procedure. Themajor components: laminin, collagen IV and entactin are conserved whilethe level of heparan sulfate proteoglycan is reduced by about 40-50%.

As used herein, the phrase “liquid cell-culture medium” generally refersto a liquid capable of growing cells and dissolving the gel-formingmaterial. The liquid cell-culture medium may comprise HEPES, or abicarbonate-based buffer, or other buffer. Optionally, it may alsocomprise serum albumin.

As used herein, the phrase “support-generating medium” refers to amixture of liquid cell-culture medium and a gel-forming material at lowdensity. The density of the gel-forming material in thesupport-generating medium is low enough that a substantial portion ofthe support-generating medium remains in liquid form (i.e. dissolved inthe liquid cell-culture medium) throughout the culture period (FIG. 5).

As used herein, the phrases “support medium” or “support matrix” refersto a thin soft gel produced spontaneously by the support-generatingmedium or by the interaction of the support-generating medium with thecultured cells, or by the interaction of the support-generating mediumwith the cell-culture surface (FIG. 3).

As used herein, the phrase “endothelial lineage” refers to endothelialcells or precursor cells or their offspring that are capable ofdifferentiating into an endothelial cell. Cells of endothelial lineageinclude, but are not limited to, endothelial progenitor cells,endothelial colony forming cells, vascular stem cells, embryonic andadult stem cells, circulating endothelial progenitor cells, angioblasts,microvasculature, arteries, veins, and lymphatic endothelial cells ofdifferent origin including but not limited to dermal, bladder, cardiac,and pulmonary.

As used herein, “viable” capillaries refer to lumenized capillariescomprising living endothelial cells.

Described herein are devices for fabrication of engineered capillarynetworks. In various embodiments, the devices comprise living cells, acell-culture surface, a support-generating medium, and a liquidcell-culture medium. In one illustrative aspect, a gel-forming materialis substantially dissolved in the liquid cell-culture medium to form asupport-generating medium, which then forms a support medium on thecell-culture surface. As used herein, “substantially dissolved”gel-forming substance means that little or no gel-forming materialremains in solid form after the gel forming-material is mixed with theliquid cell-culture medium. The support medium is formed by thesupport-generating medium or by the interaction of thesupport-generating medium with the cultured cells, or by the interactionof the support-generating medium with the cell-culture surface. Thesupport medium may partially or completely coat the cell-culturesurface. The support medium harbors the living cells. The support mediumis not rigid. The support medium permits, growth, motility, anddifferentiation of living cells. The cells may permeate the supportmedium as they grow, differentiate, and produce their own ECM andbasement membrane.

In one embodiment, the capillary fabrication device has a cell-culturesurface that is custom-patterned. In one illustrative aspect, thecell-culture surface pattern is a network-like pattern that containsregions of spatially and/or temporally variable hydrophobicities. In oneillustrative aspect, the cell-culture surface comprises hydrophobicitygradients. In one illustrative aspect, the cell-culture surface patternsare linear. In one illustrative aspect, the cell-culture surfacepatterns are nonlinear. In another illustrative aspect, the cell-culturesurface comprises both linear and non-linear patterns.

The cells of the capillary fabrication device may be of human ornon-human origin. In one embodiment, the cells are of endotheliallineage. In one embodiment, the cells comprise a plurality of cellstypes of endothelial lineage. In another embodiment, the cells compriseone or more cell types of endothelial lineage and at least oneadditional cell type not of endothelial lineage. In one aspect, thecells of non-endothelial lineage provide support and/or stabilize theforming or formed capillary networks. In one illustrative aspect, theadditional cell type is selected from the group consisting of pericytes,smooth muscle cells, fibroblasts, and any combination thereof.

In one embodiment, the cells of endothelial lineage or non-endotheliallineage are modified. As used herein, the term “modified cells”includes, but is not limited to, genetic modifications, for example, theinsertion, deletion, or change in the expression of a gene or genes in acell. The term “modified cells” also includes cells affected by anychemical, mechanical, electromagnetic or other modifications made to theculture to modify the cells. Modifications of cells may be madecontinuously or intermittently at any time before culturing the cells inthe capillary fabrication device, and/or during and/or after capillarynetwork formation.

In one embodiment, the capillary fabrication device comprises acell-culture surface having at least one hydrophobic region. In oneillustrative embodiment, the hydrophobic regions form a pattern. In oneillustrative aspect, the pattern comprises a hexagonal network of narrowbands of less hydrophilic cell-culture surface having a width of fromabout 5 microns to about 20 microns, from about 5 microns to about 15microns, from about 5 microns to about 8 microns, from about 5 micronsto about 10 microns, from about 8 microns to about 15 microns, fromabout 10 microns to about 15 microns, from about 10 microns to about 20microns, or from about 12 microns to about 20 microns; and a length offrom about 175 microns to about 300 microns, from about 200 microns toabout 300 microns, from about 175 to about 250 microns, from about 200to about 250 microns, from about 225 to about 300 microns, or from about225 to about 25 microns. These various ranges of width and length of thebands are also contemplated where the term “about” is not included. Thecustom-patterned/controlled-geometry capillaries form on top of thepredefined hexagonal network pattern of less hydrophilic cell-culturesurface.

In one embodiment, the capillary fabrication devices have a cell-culturesurface that is modified to change its hydrophobicity in certainpre-defined regions by application of a hydrophobic substance capable ofattracting the cells. Illustratively, the hydrophobic substance may beapplied by etching, stamping, contact printing, and the like.

In one embodiment, the cell-culture surface is pre-coated with a layerof a temperature sensitive polymer. The temperature sensitive polymermay become significantly dehydrated at about 37° C. and significantlyhydrated at temperatures lower than about 32° C. Illustratively, thepolymer coating is more hydrophobic in its hydrated state than in itsdehydrated state.

In one embodiment, the temperature sensitive coating is applied to thecell-culture surface in a pre-formed pattern. In another illustrativeembodiment, the temperature sensitive coating is applied to thecell-culture surface uniformly and then UV laser ablation is used topattern the coated surface by reducing the thickness of the coating,thereby reducing the hydrophobicity of that portion of the cellculture-surface. In one illustrative aspect, the temperature sensitivepolymer is poly(N-isopropylacrylamide).

In one embodiment of the capillary fabrication devices, the cell culturesurface is flat. For example, a flat surface may be a petri dish,well-plate, microscope slide, coverslip, and the like. In oneembodiment, the cell-culture surface is comprised of a material selectedfrom the group consisting of non-treated polystyrene, glass, atemperature sensitive polymer, and any combination thereof. Optionally,the cell-culture surface may contain meshes or scaffolds to furtherpattern the capillary networks in two or three dimensions.

The capillary fabrication devices may comprise any suitable liquidmedium for cell culture. Several cell-culture media are known to thoseof skill in the art. In one embodiment, the cell-culture medium is adefined medium. In one illustrative aspect, the cell-culture medium isbicarbonate based. In one illustrative aspect, the cell-culture mediumis HEPES. In one embodiment, the cell-culture medium contains one ormore serum proteins. In one illustrative aspect, the cell-culture mediumcontains serum albumin.

The gel-forming material of the capillary fabrication devices issubstantially dissolved in the liquid cell-culture medium to form thesupport-generating medium. Gel-forming materials for use in thecapillary fabrication devices are known to those of skill in the art. Inone embodiment, the gel-forming material comprises one or more ECMproteins. Extracellular matrix proteins include, but are not limited to,laminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen,TGF-β, epidermal growth factor, insulin-like growth factor, fibroblastgrowth factor, and tissue plasminogen activator, and the like. In oneillustrative embodiment, the gel-forming material of the capillaryfabrication devices is Matrigel™.

In one embodiment, the support-generating medium comprises Matrigel™diluted from about 20 to about 80 times, from about 20 to about 60times, from about 20 to about 40 times, from about 30 to about 80 times,from about 30 to about 60 times, from about 30 to about 45 times, orfrom about 45 to about 60 times in the liquid cell-culture medium. Thesevarious dilutions are also contemplated where the word “about” is notincluded.

In one embodiment, the support-generating medium of the capillaryfabrication devices comprises from about 125 μg to about 500 μg, fromabout 125 μg to about 170 μg, from about 125 μg to about 250 μg, fromabout 125 μg to about 350 μg, from about 170 μg to about 350 μg, fromabout 225 μg to about 350 μg, or from about 170 μg to about 225 μg ofECM proteins per ml of liquid cell-culture medium. These variousconcentrations are also contemplated where the word “about” is notincluded.

In the capillary fabrication devices described herein, thesupport-generating medium forms a support medium after contacting thecell-culture surface and/or the living cells. In one embodiment, theliving cells permeate the support medium and reside within the supportmedium soon after cell plating.

In one embodiment, the capillary fabrication devices comprise a supportmedium that is less than 150 microns thick. In various otherembodiments, the support medium has a thickness of from about 10 micronsto about 150 microns, about 10 microns to about 125 microns, about 10microns to about 100 microns, about 10 microns to about 75 microns,about 10 microns to about 60 microns, about 10 microns to about 50microns, about 10 microns to about 40 microns, about 20 microns to about75 microns, about 20 microns to about 60 microns, about 20 microns toabout 50 microns, about 30 microns to about 75 microns, about 30 micronsto about 60 microns, about 30 to about 50 microns, about 40 microns toabout 70 microns, or about 40 microns to about 60 microns. In anotherembodiment, the support medium has a thickness of from about 20 micronsto about 40 microns. In another embodiment, the support medium has athickness of from about 50 microns to about 100 microns. These variousthicknesses are also contemplated where the word “about” is notincluded. It is appreciated that the thickness of the support medium maynot be uniform at all portions of the cell-culture surface.

In one embodiment, a formed capillary network is described made usingthe capillary fabrication devices described herein. In one illustrativeaspect, the formed capillary network comprises tubules and issubstantially lumenized. In one illustrative embodiment, the capillarynetwork has a pre-patterned geometry. In one illustrative embodiment,the capillary network comprises tubules having a diameter of at least 5microns. In another illustrative embodiment, the capillary networkcomprises tubules having a diameter of at least 10 microns. In oneillustrative embodiment, the capillary network comprises tubules havinga diameter of at least 15 microns. In one illustrative embodiment, thecapillary network comprises tubules having a diameter of at least 20microns.

In various embodiments, the capillary fabrication devices are used togenerate forming or formed capillary networks. In one embodiment, thecapillary fabrication devices are used to assess the effects of one ormore test substances on angiogenesis. In one illustrative embodiment,the plated cells, or the cells of the forming or formed capillarynetwork are contacted with a test substance suspected to promote orinhibit angiogenesis.

In various embodiments, the ECM and basement membrane produced by theliving cells of the capillary fabrication device are detached. The ECMand basement membrane may be detached from the forming or formedcapillary networks by various methods. In one illustrative embodiment,the ECM and basement membrane are detached from the capillary network bychanging the pH of the cell-culture medium, changing the temperature ofthe cell-culture medium, contacting the capillary networks and/or ECMand basement membrane with a stream of culture medium, or anycombination thereof.

The capillary networks and/or the detached ECM and basement membrane maybe used in in vitro tissue engineering applications, for example,cell-sheet engineering, bioprinting, and the like.

The capillary networks and/or detached ECM and basement membrane mayalso be used in in vivo tissue engineering applications, for example,tissue-repair, tissue regeneration, and the like.

EXAMPLES Example 1: Sample Capillary Fabrication Devices Using Matrigel™in the Support-Generating Medium to Culture HUVECs or ECFCs

Materials.

Matrigel™ is purchased from BD Biosciences. Single-donor human umbilicalcord endothelial cells (HUVECs) are cultured according to protocolsprovided by PromoCell™. It is recommended to use HUVECs passaged lessthan 4 times. Alternatively, endothelial colony forming cells (ECFCs)are used. ECFCs may be extracted from human peripheral and umbilicalcord blood.

A stock of 1× Matrigel™ is diluted about 30 to about 60 times with about4° C. cell-culture medium (from PromoCell) to produce a homogeneoussupport-generating medium. The support-generating medium is plated innon-treated polystyrene petri dishes as the cell-culture surface whichhas a hydrophobic surface. Components of the mixture settle/stick to thebottom of the dish and form a loosely connected low density environment.About 3 ml of the support-generating medium is enough for 35 mm dishes.The support medium thickness formed on the dish is about 20 to about 40microns or when Matrigel™ is diluted about 60 times. The support mediumthickness formed on the dish is about 50 to about 100 microns or whenMatrigel™ is diluted about 30 times. The dishes are incubated for about30 to about 60 minutes in 5% CO₂ at 37° C. in a cell-culture incubator.The culture dishes containing cultured HUVECs and 35 mm dishes ofsupport-generating medium are transferred from the incubator to thecell-culture hood at the same time to minimize heat shock.

After harvesting the cultured HUVECs, the cells are added to the dishescontaining the support-generating medium at about 50 to about 300cells/mm² to complete the capillary fabrication device. The capillaryfabrication device is then incubated in 5% CO₂ at 37° C. To produce afully connected network, cell densities higher than 150 cells/mm² areused. To distribute the plated HUVECs uniformly, the dishes are gentlyswirled alternately clockwise and counterclockwise. Thesupport-generating medium is exchanged about every 48 hours with freshpre-warmed support-generating medium, prepared as above. If thesupport-generating medium is removed (for example, after about 60minutes of incubation at 37° C.), and not replenished withsupport-generating medium, then the HUVECs do not form lumenizedcapillary networks. After formation of capillary networks, replacing thesupport-generating medium with regular liquid cell-culture medium maykeep the capillaries viable, but formation of new capillaries from cellsnot integrated in capillaries, may be significantly reduced.

Optionally, the suspended cells may be mixed with the support-generatingmedium immediately before cell plating (keeping the dilution ratio ofMatrigel™ in the support-generating medium between about 1:30 and about1:60). This procedure eliminates the step of incubating the dishes forabout 30 to about 60 minutes and is suited to high-throughputexperiments.

Other sources of modified or unmodified cells may be used, includingthose from human and non-human sources. Endothelial lineage cells mayalso be used.

Optionally, the cell culture surface may be a flat surface, includingbut not limited to a well-plate, slide, or coverslip.

A glass culture surface, such as a slide or coverslip, allows forimproved high-resolution imaging of the lumenized capillary networksusing an inverted or upright microscope for assaying chemical treatmentsto promote or inhibit vascularization or other purposes.

Optionally, other support/stabilizing cells can be added to the cellculture, including but not limited to pericytes, smooth muscle cells,fibroblasts, myoblasts or any other cell types capable of interactingwith or stabilizing the forming or formed lumenized capillary network.

Optionally, modifications to the cells, support-generating medium, orcell-culture surface can be made continuously or intermittently at anytime before culturing the cells in the capillary fabrication device,during culturing in the capillary fabrication device and during andafter capillary formation and during any subsequent applications. Suchmodifications are useful in tissue engineering and tissue repairapplications which require functional lumenized capillary networks.

Example 2: Sample Custom-Patterned/Controlled-Geometry CapillaryFabrication Devices

In this example, the cell-culture surface in the capillary fabricationdevice has hydrophobicity gradients. For example, the non-treatedpolystyrene petri dish in Example 1 can be replaced with a patternedultra low adhesive polystyrene dish before plating support-generatingmedium. The ultra low adhesive polystyrene dish provides a cell-culturesurface which is more hydrophilic than regular non-treated polystyrenepetri dishes. The pattern on the ultra low adhesive polystyrene dishcomprises a hexagonal network of narrow bands of 5 to 15 microns inwidth and 200 to 250 microns in length of less hydrophilic cell-culturesurface. Cells of endothelial lineage can be used. Thecustom-patterned/controlled-geometry capillaries form on top of thepredefined hexagonal network pattern of less hydrophilic cell-culturesurface.

Optionally, the non-treated polystyrene cell-culture surface in Example1 can be pre-coated with a layer of temperature sensitive polymers, likepoly(N-isopropylacrylamide). The temperature sensitive polymer isdehydrated at 37° C. and hydrated at temperatures lower than 32° C. Thepolymer coating is more hydrophobic in its hydrated state. The coatinghydrophobicity also depends on the thickness of the coating. A coatingthickness of more than about 3 nm results in a hydrophobic cell-culturesurface at 37° C. The non-treated polystyrene is coated uniformly andthen UV laser ablation used to pattern the coated surface by reducingthe thickness of the coating according to a customized pattern. Thecells in the custom-patterned capillary fabrication device formlumenized capillaries over areas with thinner coating. The cell-culturesurface may also be modified by methods including, but not limited to,etching, stamping, and contact printing.

Use of a temperature sensitive polymer in this protocol allowsdetachment of the formed capillary network and the associated ECM andbasement membrane with minimal damage. The cell-culture temperature canbe adjusted to facilitate detachment. The detached capillary network andthe associated ECM and basement membrane can be used in tissueengineering and tissue repair applications.

The custom-patterned capillary fabrication device is capable ofmanufacturing capillary networks of specified pattern using cell-culturesurfaces with network patterns consisting of narrow bands of 5 to 15microns in width and specified length and connectivity. The device iscapable of producing replicas of 2-dimensional capillary networks likechoroidal capillaries in the retina.

Example 3: Sample Capillary Assay Devices

Capillary networks are grown as described in Example 1 using thecapillary fabrication device or Example 2 using the custom-patternedcapillary fabrication device combined with one or modifications with theaim of determining the effect of those modifications on the culturedcells or on the resulting capillary networks.

The capillary assay device can be used for determining the proangiogeniceffects of different isoforms of vascular endothelial growth factor(VEGF) including soluble and ECM-bound isoforms. The cells in thecapillary assay device are cultured inside the support medium, thus thedevice provides quasi-in vivo conditions for the interaction of VEGFisoforms and the cells. Cells in the 2-dimensional standard Matrigel™capillary fabrication device are cultured on top of a thick solidifiedgel which differs significantly from in vivo conditions.

The capillary assay device also can be used for determining the effectsof various toxic materials such as, for example, lead, arsenic, and thelike on angionesis. Further, the capillary assay device may be used tostudy cellular mechanosignaling related to angiogenesis, or the pro/antiangiogenic effects of mechanical stressors such as, for example,ultrasonic waves or altered pressure. Additionally, the capillary assaydevice may be used to study the pro/anti angiogenic effects of magneticor electric fields, or electromagnetic waves.

Example 4: Engineered Capillary Networks with Modified Cells

Cells used in this example can be modified before, during or aftergrowth of capillary network using the capillary fabrication device(Example 1) or the custom-patterned capillary fabrication device(Example 2). After a sufficient period of growth, the capillaries aredetached in a manner causing minimal or no damage to the capillarynetwork and can be used for in vivo or in vitro implantation.

The capillary networks formed using custom-patterned capillaryfabrication device (Example 2) have predefined geometry which makes themsuitable for tissue engineering and repair applications with specifiedgeometries and metabolic rates.

In vitro modification of cells used to grow the capillary networks maybe useful in minimizing the chance of rejection in allotransplantationor xenotransplantation.

Example 5: Engineered ECM and/or Basement Membrane

Capillary networks are grown as described in Example 1 using thecapillary fabrication device or Example 2 using the custom-patternedcapillary fabrication device. After a sufficient period of growth, thecells are destroyed, for example, by washing the capillary network witha liquid, for example, distilled water, while retaining the ECM andbasement membrane. Then the patterned ECM and basement membrane aredetached in a manner causing minimal or no damage to the capillarynetwork, for example, by using a stream of culture media, or by usingtemperature sensitive polymers. Adjusting the temperature or pH tomodify hydrophobicity can facilitate detachment. For example, adjustingthe pH of the culture media to slightly acidic (pH˜6-6.5) is found tofacilitate detachment of ECM.

The thickness of the ECM may be controlled by the concentration of thesupport-generating medium. For example, using diluted Matrigel™ as agel-forming material, the thickness of manufactured ECM is about 20 to40 microns when Matrigel™ is diluted at a ratio of 1:60 and about 50 to100 microns when diluted at a ratio of 1:30. The ECM has a net-work likemorphology. The support matrix is organized in cables that are about 50to 500 microns long when cultured for 5 days using a 1:30 dilution ofMatrigel™. The width of the cables is about 15 to 100 microns. Thelength of the cables increases over the duration of the culture period.For example, cables about 1.5 mm long are observed after 10 days ofculture. At more dilute concentrations (e.g. Matrigel™ at 1:60) thecables elongate faster. Thus, the dilution of the gel forming materialand duration of culture can be controlled to generate ECM withcustomized morphology.

The cell-remodeled basement membrane and ECM may then be used to enhancegrowth/regrowth of vasculature in vivo or in vitro. In vivo implantationcan be examined.

After in vivo implantation of the cultured ECM and/or basement membrane,regrowth of capillaries and pericyte recruitment occurs as cells invadethe empty sleeves of basement membrane and/or ECM left behind by thedestroyed capillaries. Since extracellular components are highlyconserved, this organically-manufactured basement membrane/ECM is usefulfor medical tissue-repair applications with minimal transfer of externalfactors and thus significant reduction of the likelihood of rejectionafter implant.

The engineered ECM and basement membrane using custom-patternedcapillary fabrication devices (Example 2) have predefined geometry whichmakes them suitable for growth/regrowth of vasculature in tissueengineering and tissue repair applications with specified geometries andmetabolic rates.

Example 6: Use of Engineered ECM and Basement Membrane in Cell-SheetEngineering and Bioprinting

In this example quasi-two-dimensional layers of engineered ECM andbasement membrane (Example 5) are placed in horizontal layers separatedby 100 to 200 micron layers of engineered tissue in cell-sheetengineering and bioprinting applications. The resulting enhanced growthof capillary networks in cell-sheet engineering and bioprintingapplications results in a functional vascular network to provide bloodflow to all volumes of the engineered tissue, enhancing the likelihoodof integration of engineered 3-dimensional tissues.

Example 7: Use of Implantable Capillary Networks in Cell-SheetEngineering and Bioprinting

In this example quasi-two-dimensional layers of formed or formingcapillary networks and associated ECM and basement membrane (Example 4)formed using the capillary fabrication device or custom-patternedfabrication device are placed in horizontal layers separated by 100 to200 micron layers of engineered tissue in cell-sheet engineering andbioprinting applications. Integration of the capillary networks withexisting or engineered larger vessels in these applications results in afunctional vascular network to provide blood flow to all volumes of theengineered tissue, enhancing the likelihood of integration of engineered3-dimensional tissues.

Example 8: Formation and Patterning of Hydrophobic Domains on the CellCulture Surface

Highly hydrophobic domains in the culture surface coating are generatedwith polydimethylsiloxane (PDMS) which the cells do not populate.Moderately hydrophobic domains, which cells invade and populate, aregenerated with long carbon chain silanes, for exampletrichloro(octyl)silane, trichloro(decyl)silane,trichloro(dodecyl)silane, trichloro(octadecyl)silane,trimethoxy(octyl)silane, trimethoxy(decyl)silane,trimethoxy(dodecyl)silane, trimethoxy(octadecyl)silane,triethoxy(octyl)silane, triethoxy(decyl)silane,triethoxy(dodecyl)silane, or triethoxy(octadecyl)silane. Photoresistmasking may be used to generate patterns of moderately hydrophobic andhighly hydrophobic domains on the cell culture surface.

For example, a clean glass slide is functionalized with one or more longcarbon silanes (moderately hydrophobic) to create a carbon chainmonolayer. The slide is exposed in a 0.2-2% silane in organic solvents(e.g. methanol, acetone, toluene, heptane, carbon tetrachloride, xylene,and the like) followed by a methanol rinse and then dried under nitrogengas. The surface is then spin-coated with a thin, approximately 5-10 μmlayer of positive photoresist (e.g. SPR220, PMMA, and the like). Aftersoftbaking the substrate is exposed to UV light through a mask with aspatial pattern to be replicated by the cells of the functionalizedglass substrates. After removing the exposed photoresist a thin layer ofPDMS is spin-coated on. The PDMS is then etched by tetrabutylammoniumfluoride (TBAF) solution in tetrahydrofuran (THF) until the layerbecomes 2-5 μm thick. The substrates are then briefly exposed to UVlight without any mask. The photoresist domains masked during the priorexposure are removed, revealing the silane coated glass domainssurrounded by PDMS domains.

Example 9: HUVECs do not Invade Highly Hydrophobic PDMS Domains

A capillary fabrication device is utilized to culture HUVECs asdescribed in Example 1. A PDMS coating is applied as described inExample 8 to a polystyrene surface. The HUVECs grow and form capillarieson a polstyrene surface containing a support medium, but do not grow onregions of the surface coated with PDMS (FIG. 7).

Example 10: Implantation of Viable Capillary Networks

Capillary networks formed using the capillary fabrication devices areimplanted ex ovo. Chicken eggs are incubated for 3 days at 37° C. On day3, eggs are broken and embryos are transferred to 10 cm dishes. Theembryos are grown for 3-4 more days. On day 6-7, capillary networksformed by HUVECs are implanted in up to four regions of chorioallantoicmembrane (CAM). The CAMs are scratched with a sharp needle until a smallamount of blood appears. The capillaries are then injected into the CAMusing a micro-pipette.

The capillary networks are labeled with Calcein AM to examine theirviability. Calcein AM is used as an indicator of viability because itdiffuses out of cells when they die. Implanted capillary networks areviewed under microscope after implantation. Viable implanted HUVEC cellsare observed 2 days after implantation.

While the invention has been illustrated and described in detail in theforegoing description, such an illustration and description is to beconsidered as exemplary and not restrictive in character, it beingunderstood that only the illustrative embodiments have been describedand that all changes and modifications that come within the scope of theinvention are desired to be protected. Those of ordinary skill in theart may readily devise their own implementations that incorporate one ormore of the features described herein, and thus fall within the scope ofthe present invention.

What is claimed is:
 1. A device for fabrication of engineered capillarynetworks comprising: endothelial lineage cells; a non-treatedpolystyrene cell-culture surface; and a low-density support-generatingmedium, the low-density support-generating medium comprising a gelforming material and a liquid cell-culture medium in a ratio of 1:30 to1:60, wherein the gel forming material is substantially dissolved in thecell-culture medium and forms a support medium having a thickness ofbetween 20 microns and 100 microns, and wherein the support mediumcovers the endothelial lineage cells and the cell-culture medium.
 2. Thedevice of claim 1, wherein the endothelial lineage cells are selectedfrom the group consisting of stem cells, endothelial progenitor cells,circulating endothelial cells, and lymphatic endothelial cells.
 3. Thedevice of claim 1, further comprising at least one additional cell typeselected from the group consisting of pericytes, smooth muscle cells,fibroblasts, and any combination thereof.
 4. The device of claim 1,wherein the non-treated polystyrene cell-culture surface comprises atleast one hydrophobic region.
 5. The device of claim 1, wherein thenon-treated polystyrene cell-culture surface comprises a coating of atleast one temperature sensitive polymer.
 6. The device of claim 1,wherein the non-treated polystyrene cell-culture surface is modified byetching, stamping, contact printing, UV laser ablation, or anycombination thereof.
 7. The device of claim 1, wherein the liquidcell-culture medium comprises one or more components selected from thegroup consisting of serum albumin, a bicarbonate-base, a HEPES buffer,and an extracellular matrix (ECM) protein.
 8. The device of claim 1,wherein the gel forming material comprises at least one protein selectedfrom the group consisting of laminin, collagen IV, heparan sulfateproteoglycans, entactin/nidogen, TGF-β, epidermal growth factor,insulin-like growth factor, fibroblast growth factor, and tissueplasminogen activator.
 9. The device of claim 1, wherein the gel-formingmaterial is dissolved in the liquid cell-culture medium to yield an ECMprotein concentration of from 170 μg ECM proteins per ml of liquidcell-culture medium to 350 μg ECM proteins per ml of liquid cell-culturemedium.
 10. The device of claim 1, wherein the support medium harborsthe endothelial lineage cells.
 11. The device of claim 1, wherein thesupport medium having a thickness of between 20 microns and 100 micronson the non-treated polystyrene cell-culture surface.
 12. The device ofclaim 1, wherein the support medium has a thickness from 20 microns to40 microns on the non-treated polystyrene cell-culture surface.
 13. Thedevice of claim 1, wherein the non-treated polystyrene cell-culturesurface comprises a network-like pattern containing regions of varyinghydrophobicity.
 14. The device of claim 1, wherein the gel formingmaterial is MATRIGEL.
 15. The device of claim 1, wherein the gel formingmaterial and the liquid cell-culture medium are in a ratio of 1:30 to1:60, and gel forming material is MATRIGEL.
 16. The device of claim 1,wherein the gel forming material and the liquid cell-culture medium arein a ratio of 1:30 to 1:60, the support medium has a thickness from 20microns to 40 microns on the non-treated polystyrene cell-culturesurface, and gel forming material is MATRIGEL.
 17. The device of claim1, wherein the gel forming material and the liquid cell-culture mediumare in a ratio of 1:30 to 1:60, the support medium has a thickness from20 microns to 40 microns on the non-treated polystyrene cell-culturesurface, the non-treated polystyrene cell-culture surface comprises anetwork-like pattern containing regions of varying hydrophobicity, andgel forming material is MATRIGEL.